Listeria monocytogenes (Lm) is a Gram-positive intracellular bacterial pathogen capable of invading numerous host cell types. Lm infections can lead to severe disease in humans and most often affects immunocompromised individuals, pregnant women, and the elderly. Of particular concern is the ability of Lm to invade the central nervous system (CNS), leading to life-threatening meningitis and encephalitis. The identity of factors necessary to facilitate Lm brain infection has remained unclear. We have discovered that an Lm surface protein, InlF, is required for successful colonization of the brain in mice. Moreover, the results of affinity chromatography and mass spectrometry analysis indicate that InlF interacts with the brain endothelial cell surface protein, Vimentin, splicing factor Sfpq and nucleoprotein AHNAK. The focus of this proposal is to elucidate how InlF mediates Lm infection of the brain. We hypothesize that InlF interacts with Vimentin, accompanied by Sfpq and AHNAK, to mediate entry of Lm into brain cells and that in the absence of Vimentin, Lm infection of the brain will be severely compromised in vivo due to the importance of InlF-Vimentin interactions for successful brain colonization. We further hypothesize that InlF is required for passage across the BBB to establish an Lm infection in the brain. In Aim I, gentamicin protection assays will be used to determine the requirement of Vimentin for InlF-mediated invasion of cultured cell lines. Fluorescence microscopy with Lm strains expressing green fluorescent protein (GFP) and InlF-coated green-fluorescent beads will be used to more directly examine the ability of InlF to interact with host cells. In addition, a biochemical approach will be taken to demonstrate direct protein binding and identify the regions involved in the InlF-Vimentin protein-protein interaction. In Aim II, the contribution of InlF-Vimentin interactions to Lm infection in vivo will be determine by infection studies in Vimentin or AHNAK knockout mice. In vitro infection assays and fluorescence microscopy will be performed with primary brain cells cultured from normal and knockout mice to determine if InlF mediates infection of distinct primary brain cell types and the importance of Vimentin and AHNAK for infection. In Aim III, we will determine if InlF mediates BBB passage by direct invasion or cell-to-cell spread. Gentamicin protection assays with primary mouse brain microvascular endothelial cells and brain endothelial cell lines will be performed to determine if InlF is required for direct invasion of cells that constitute the BBB. Heterologous spread assays with Lm infected monocytes and brain endothelial cells will be used to assess the importance of InlF in cell-to-cell spread into the brain. Finally, infection of mice with cell-to-cell spread defective actA or actA/inlF Lm strains will determine the in vivo contribution of InlF for colonization of the brain by direct invasion versus cell-to-cell spread. Te proposed studies will provide insights into the protein-protein interactions facilitating Lm invasin of the brain and may identify novel targets for preventing infections of the brain by Lm and other intracellular pathogens.